The usefulness of multiplex polymerase chain reaction was evaluated and fluorescent detection with 20 microsatellites was used for paternity testing of Liangshan semi-fine wool sheep. The results in Cervus 2.0 indicated that the combined exclusion probability was 0.998666 when parents were both unknown. If father was known, combined exclusion probability was 0.999994, with confidence level of 95%. Finally, the biological parents of 215 individuals in offspring were found from eight candidate fathers and 147 candidate mothers, and a mixed pedigree was constructed. It is useful for linkage analysis of sheep chromosomes and QTL location, also for the study of population structure, history, and diversity.